Comparison of intensity versus lifetime-based approaches for quantitative in vivo FRET imaging

Fluorescence Resonance Energy Transfer (FRET) enables measurement of the distance separating two light-sensitive molecules. The method has been employed in countless biological applications to monitor inter and intra-molecular interactions and conformations in the 1-10 nm range. Importantly, FRET measurements can be performed with both ratiometric intensity FRET (ratiometric FRET) and fluorescence lifetime imaging-based FRET (FLI-FRET) approaches. Although FRET has long been used for in vitro microscopy, using either the intensity- or lifetime-based approaches, it has recently been extended to in vivo application, for instance to quantify drug-target engagement in animal models of cancer. The in vivo method had thus far only been demonstrated using macroscopic FLI-FRET (MFLI-FRET). Herein, we present a comparison of FRET quantification in vivo using either ratiometric FRET (3-cube, IVIS system) and MFLI-FRET (time-resolved ICCD system) in order to demonstrate the superiority of the latter for in vivo applications.